REACTIVITY | SENSITIVITY | MW (kDa) | SOURCE |
---|---|---|---|
H M R All | Endogenous | Rabbit |
Western blot analysis of extracts from Jurkat cells, untreated or calyculin A-treated (0.1 µM for 30 minutes prior to lysis), using Phospho-(Ser/Thr) Akt Substrate Antibody. Proteins were separated by 2-D electrophoresis prior to blotting.
Learn more about how we get our images.Western blot analysis of extracts from A431 cells, untreated or calyculin A-treated, using Phospho-(Ser/Thr) Akt Substrate Antibody.
Learn more about how we get our images.Western blot analysis of extracts from C2C12 cells, untreated or insulin-treated (100 nM for 15 minutes prior to lysis), using Phospho-(Ser/Thr) Akt Substrate Antibody.
Learn more about how we get our images.Immunohistochemical analysis of paraffin-embedded human breast carcinoma control (left) or lambda phosphatase-treated (right), using Phospho-(Ser/Thr) Akt Substrate Antibody.
Learn more about how we get our images.Immunohistochemical analysis of paraffin-embedded human colon carcinoma, using Phospho-(Ser/Thr) Akt Substrate Antibody.
Learn more about how we get our images.Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using Phospho-(Ser/Thr) Akt Substrate Antibody.
Learn more about how we get our images.For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Loading of prestained molecular weight markers (#13953, 5 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity utilizing Protein A magnetic separation.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
10X Cell Lysis Buffer: (#9803) To prepare 10 ml of 1X cell lysis buffer, add 1 ml cell lysis buffer to 9 ml dH2O, mix.
NOTE: Add 1 mM PMSF (#8553) immediately prior to use.
A cell lysate pre-clearing step is highly recommended to reduce non-specific protein binding to the Protein A Magnetic beads. Pre-clear enough lysate for test samples and isotype controls.
IMPORTANT: Pre-wash #73778 magnetic beads just prior to use:
Carefully remove the buffer once the solution is clear. Add 500 μl of 1X cell lysis buffer to the magnetic bead pellet, briefly vortex to wash the beads. Place tube back in magnetic separation rack. Remove buffer once solution is clear. Repeat washing step once more.
IMPORTANT: The optimal lysate concentration will depend on the expression level of the protein of interest. A starting concentration between 250 μg/ml-1.0 mg/ml is recommended.
IMPORTANT: Appropriate isotype controls are highly recommended in order to show specific binding in your primary antibody immunoprecipitation. Use Normal Rabbit IgG #2729 for rabbit polyclonal primary antibodies, Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 for rabbit monoclonal primary antibodies, and Mouse (G3A1) mAb IgG1 Isotype Control #5415 for mouse monoclonal primary antibodies. Isotype controls should be concentration matched and run alongside the primary antibody samples
Proceed to one of the following specific set of steps.
NOTE: To minimize masking caused by denatured IgG heavy chains (~50 kDa), we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (#45262) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127). To minimize masking caused by denatured IgG light chains (~25 kDa), we recommend using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127).
posted December 2008
revised April 2018
Protocol Id: 410
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: Do not allow slides to dry at any time during this procedure.
For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
posted February 2010
revised March 2016
Protocol Id: 300
Application | Dilutions |
---|---|
Western Blotting | 1:1000 |
Immunoprecipitation | 1:50 |
Immunohistochemistry (Paraffin) | 1:500 |
Peptide ELISA (DELFIA) | 1:500 |
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
Phospho-(Ser/Thr) Akt Substrate Antibody preferentially recognizes peptides and proteins containing phospho-Ser/Thr preceded by Lys/Arg at positions -5 and -3, in a manner largely independent of other surrounding amino acids. Some cross-reactivity is observed for peptides that contain phospho-Ser/Thr preceded by Arg/Lys at positions -3 and -2. No cross-reactivity is observed with the corresponding nonphosphorylated sequences or with other phospho-Ser/Thr-containing motifs. By ELISA, the antibody recognizes a wide range of phosphorylated Akt substrate peptides, and, by 2-D gel Western blot analysis, it recognizes a large number of proteins presumed to be Akt substrates.
Human, Mouse, Rat, All Species Expected
Polyclonal antibodies are produced by immunizing animals with phospho-Akt substrate peptides . Antibodies are purified by protein A and peptide affinity chromatography.
An important class of kinases, referred to as Arg-directed kinases or AGC-family kinases, includes cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), protein kinase C, Akt, and RSK. These kinases share a substrate specificity characterized by Arg at position -3 relative to the phosphorylated Ser or Thr (1,2). Akt plays a central role in mediating critical cellular responses including cell growth and survival, angiogenesis, and transcriptional regulation (3-5). While a number of Akt substrates are known (such as GSK-3, Bad, and caspase-9) many important substrates await discovery. Akt phosphorylates substrates only at Ser/Thr in a conserved motif characterized by Arg at positions -5 and -3 (6). Phospho-Akt substrate-specific antibodies from Cell Signaling Technology are powerful tools for investigating the regulation of phosphorylation by Akt and other Arg-directed kinases, as well as for high throughput kinase drug discovery.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. Use of Cell Signaling Technology (CST) Motif Antibodies within certain methods (e.g., U.S. Patents No. 7,198,896 and 7,300,753) may require a license from CST. For information regarding academic licensing terms please have your technology transfer office contact CST Legal Department at CST_ip@cellsignal.com. For information regarding commercial licensing terms please contact CST Pharma Services Department at ptmscan@cellsignal.com.
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Product # | Size | Price |
---|---|---|
9611S | 100 µl | N/A |
9611L | 300 µl | N/A |
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